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United States Patent |
6,210,953
|
Osman
,   et al.
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April 3, 2001
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Bacillus thuringiensis isolate BTC-18 with broad spectrum activity
Abstract
A broad spectrum Bacillus thuringiensis strain, BtC-18, is provided which
displays pesticidal activity against nematodes and against insects from
the orders Lepidoptera, Diptera and Coleoptera.
Inventors:
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Osman; Yehia A. (Mansoura, EG);
Madkour; Magdy A. (Giza, EG);
Bulla, Jr.; Lee A. (Laramie, WY)
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Assignee:
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University of Wyoming (Laramie, WY);
Agricultural Genetic Engineering Research Institute (Giza, EG)
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Appl. No.:
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218931 |
Filed:
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December 22, 1998 |
Current U.S. Class: |
435/252.5 |
Intern'l Class: |
C12N 001/20 |
Field of Search: |
435/252.5
|
References Cited
U.S. Patent Documents
4948734 | Aug., 1990 | Edwards et al. | 435/252.
|
5080897 | Jan., 1992 | Gonzalez et al. | 424/93.
|
Foreign Patent Documents |
0 537 105 A1 | Apr., 1993 | EP.
| |
0537105 | Apr., 1993 | EP.
| |
WO 95/02693 | Jan., 1995 | WO.
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WO 97/12980 | Apr., 1997 | WO.
| |
Other References
Chestukhina GG, et al., "Bacillus thuringiensis ssp. galleriae
simultaneously produces two gamma-endotoxins differing strongly in primary
structure and entomocidal activity." FEBS. Lett. 232: 249-251, May 1988.*
Yan B, et al. "Protein components and toxicity of delta-endotoxin crystals
from Bacillus thuringiensis Tm 13-14." Kunchong Xuebao 38: 138-145
(Abstract only), 1995.*
Schnepf HE, et al. (1995) "Bacillus Thuringiensos Toxins: Regulation,
Activities, and Structural Diversity" Curr. Opinion Biotech. 6: 305-312.
Shin, et al. "Distribution of cryV-Type Insectiial Protein Genes Bacillus
thuringiensis and Cloning of cryv-Type Genes from Bacillus thuringiensis
subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus", Applied
and Enviromental Microbiology, Jun. 1995, p. 2402-2407, vol. 61, No. 6.
Chambers, et al., "Isolation and Characterization of a Novel Insecticdial
Crystal Protein Gene from Bacillus thuringiensis subsp. aizawai " ,
Journal of Bacteriology, Jul. 1991, p. 3966-3976, vol. 173, No. 13.
Lee, et al., "Diversity of Protein Inclusion Bodies and Identification of
Mosquitocidal Protein in Bacillus thuringiensis subsp. Israelensis ",
Biochemical and Biophysical Research Communications, Jan. 31, 1985, p.
953-960, vol. 126, No. 2.
Shin et al. "Distribution of CryV-type Insectcdial Protein Genes in
Bacillus Thuringiensis and cloning of CryV-type Genes from Bacillus
Thuringiensis subp. Kurstaki and Bacillus Thuringiensis subsp.
Entomocidus", Applied and Environmental Microbiology 61:2402-2407 (1995).
Chambers et al. Isolation and Characterization of a Novel Insecticdial
Crystal Protein Gene from Bacillus Thuringiensis subsp. Aizawai, Journal
of Bacteriology 173:3966-3976 (1991).
Lee et al. Diversity of Protein Inclusion Bodies and Idenfication of
Mosquitocidal Protein in Bacillus Thuringiensis subsp. israelensis ,
Biochemical and Biophysical Research Communications 12:953-960 (1985).
|
Primary Examiner: Nelson; Amy J.
Attorney, Agent or Firm: Alston & Bird LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of application Ser. No.
09/003,217, filed Jan. 6, 1998, now U.S. Pat. No. 5,986,177, which claims
the benefit of Provisional Application Serial No. 60/035,361, filed Jan.
10, 1997, the disclosures of which are herein incorporated by reference.
Claims
What is claimed is:
1. A biologically pure culture of a Bacillus thuringiensis strain having
pesticidal activity against nematodes and against insects from the orders
Lepidoptera, Diptera and Coleoptera, wherein said strain is BtC-18
deposited as ATCC Accession No. 55922, or a mutant thereof.
2. A pesticidal composition comprising a Bacillus thuringiensis strain
having pesticidal activity against nematodes and against insects from the
orders Lepidoptera, Diptera and Coleoptera, wherein said strain is BtC-18
deposited as ATCC Accession No. 55922, or a mutant thereof.
3. The pesticidal composition of claim 2, further comprising at least one
member selected from the group consisting of carriers, surfactants,
adjuvants, fertilizers, micronutrient donors, herbicides, insecticides,
fungicides, bacteriocides, nematocides, and mollusocides.
4. The pesticidal composition of claim 2, wherein said pesticidal activity
comprises activity against rootworm.
5. A method for killing pests comprising applying a pesticidal composition
to the environment of at least one target pest, said pesticidal
composition comprising a Bacillus thuringiensis strain having pesticidal
activity against nematodes and against insects from the orders
Lepidoptera, Diptera and Coleoptera, wherein said strain is BtC-18
deposited as ATCC Accession No. 55922, or a mutant thereof.
6. The method of claim 5, wherein said pesticidal activity comprises
activity against rootworm.
7. The method of claim 5, wherein said pesticidal composition further
comprises at least one member selected from the group consisting of
carriers, surfactants, adjuvants, fertilizers, micronutrient donors,
herbicides, insecticides, fungicides, bacteriocides, nematocides, and
mollusocides.
8. The method of claim 5, wherein said applying is by an application method
selected from the group consisting of leaf application, seed coating, and
soil application.
Description
FIELD OF THE INVENTION
The present invention relates to novel Bacillus thuringiensis strains, to
novel toxin genes, to the proteins encoded by the genes, and to the use of
genes and proteins.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis is a gram-positive soil bacterium characterized by
its ability to produce crystalline inclusions during sporulation. The
crystalline inclusions can, in some subspecies, account for 20 to 30
percent of the dry weight of the sporulated cell and may be composed of
more than one protein. Crystals are composed primarily of a single
polypeptide, a protoxin, which may also be a component of the spore coat.
The protoxin genes are located mainly on large plasmids, although
chromosomally encoded endotoxins have been reported.
The crystal proteins exhibit a highly specific insecticidal activity. Many
B. thuringiensis strains with different insect host spectra have been
identified. They are classified into different serotypes or subspecies
based on their flagellar antigens.
The protoxin does not exhibit its insecticidal activity until after oral
intake of the crystalline body. The crystal is dissolved in the intestinal
juice of the target insects. In most cases, the actual target component
(toxin) is released from the protoxin as a result of proteolytic cleavage
caused by the action of proteases from the digestive tract of the insects.
The activated toxin interacts with the midgut epithelium cells of
susceptible insects.
Electrophysiological and biochemical evidence suggests that the toxins
generate pores in the cell membrane, thus disrupting the osmotic balance.
Consequently, the cells swell and lyse. For several B. thuringensis
toxins, specific high-affinity binding sites have been demonstrated to
exist on the midgut epithelium of susceptible insects. Nucleotide
sequences have been recorded for a large number of B. thuringiensis (Bt)
crystal protein genes. Several sequences are nearly identical, and have
been designated as variations of the same gene. The crystal protein (Cry)
genes specify a family of related insecticidal proteins. The genes are
divided into major classes and subclasses characterized by both the
structural similarities and the insecticidal spectra of the encoded
proteins. The classification, explained by Hofte and Whiteley (1989)
Microbiol. Rev., 53:242-255, placed the known insecticidal crystal
proteins into four major classes. The four major classes were
Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II),
Coleoptera-specific (III), and Diptera-specific (IV) genes. Additional
classes have since been added.
The Cry1 genes are undoubtedly the best-studied crystal proteins. The Cry1
proteins are typically produced as 130 to 140 kDa protoxin proteins which
are proteolitically cleaved to produce active toxin proteins about 60 to
70 Kda. The active portion or toxic domain is localized in the N-terminal
half of the protoxin. Six groups of Cry1 proteins were known in 1989 when
the Hofte and Whiteley article was published. These groups were designated
IA(a), IA(b), IA(c), IB, IC, and ID. Since 1989, additional proteins have
been discovered and classified as Cry1E, Cry1F, Cry1G, Cry1H, and Cry1X.
The spectrum of insecticidal activity of an individual protoxin from Bt
tends to be quite narrow. That is, a given crystal protein is active
against only a few insects. None of the crystal proteins active against
Coleopteran larvae such as colorado potato beetle (Leptinotarsa
decemlineata) or yellow mealworm (Tenebrio molitor) have demonstrated
significant effects on members of the genous Diabrotica particularly D.
virgifera virgifera, the western corn rootworm (WCRW) or D. longicornis
barberi, the northern corn rootworm.
Insect pests are a major factor in the loss of the world's commercially
important agricultural crops. Broad spectrum chemical pesticides have been
used extensively to control or eradicate pests of agricultural importance.
However, there is substantial interest in developing effective alternative
pesticides.
Microbial pesticides have played an important role as alternatives to
chemical pest control. The most extensively used microbial product is
based on the bacterium Bacillus thuringiensis. However, as noted above,
the majority of Bt strains have a narrow range of activity. There is
therefore needed microbial strains with a broad range of insecticidal
activity for use as broad spectrum insecticides and as a source for
additional toxin genes and proteins.
SUMMARY OF THE INVENTION
A broad spectrum Bacillus strain is provided. The strain is active against
insects from at least the orders Lepidoptera, Diptera, and Coleoptera.
Additionally, the strain is active against nematodes, and rootworms.
Disclosed in this invention is the isolation and partial purification of
the crystal protein complex from this strain. The crystal protein complex
is demonstrated to be active against rootworms and other pests. Genes,
proteins, and their use, as well as the use of the strain are provided.
The complete nucleotide sequence of a Cry5-like gene herein designated as
Cry1I which was isolated from this strain is also provided.
The methods and compositions of the invention may be used in a variety of
systems for controlling pests, particularly plant pests.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B: FIG. 1A shows an electron micrograph with a light micrograph
insert; FIG. 1B shows an electron micrograph with the different kinds of
crystal shapes as indicated by the arrows or arrow heads. The three types
of crystal shapes may correlate with the three kinds of insecticidal
activity toward at least three insect orders, namely: Lepidoptera, Diptera
and Coleoptera.
FIG. 2: Plasmid profile of BtC-18. Lane a: DNA molecular weight standards;
Lane b: B. thuringiensis subsp. kurstaki; Lane c: Bacillus thuringiensis
subsp. aizawia; and, Lane d: strain C-18. BtC-18 also has different
plasmid DNA profiles from the other known B. thuringiensis subspecies,
such as subspecies israelensis and tenebrionis, (data not shown).
FIGS. 3A-3C: Protective effect of BtC-18 against the root nematode
Meloidogyne incognita. In panel A: the roots of infected tomato plant with
the nematode (positive control). panel B: the roots of tomato plants
infected with nematode eggs before BtC-18 was applied, and panel C: roots
of plant treated first with BtC-18 and then infected with nematode eggs.
FIGS. 4A-4B: PCR product profile of BtC-18. In FIG. 4A: Lane a contains DNA
molecular weight standards; Lane b, B. thuringiensis subsp. kurstaki; Lane
c, BtC-18; Lane d: B. thuringiensis subsp. israelensis; Lane e, BtC-18. In
FIG. 4B: Lane a contains DNA standards; Lane f, Bacillus thuringiensis
subsp. tenebrionis; Lane g, BtC-18. The results show that BtC-18 produced
the same PCR product profile as B. thuringiensis subsp. kurstaki, but
different PCR products with Dipteran and Coleopteran DNA primers.
FIGS. 5A-5E. Nucleotide and amino acid sequence of the Cry1I gene isolated
from BtC-18.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for controlling plant pests are provided. In
particular, novel broad spectrum Bacillus strains having a wide range of
insecticidal activity are provided. The strains are useful as insecticidal
agents. In addition, the crystal protein complex from one of these strains
has been purified and is shown to have insecticidal properties. Methods of
its purification are discussed and evidence of its activity against
rootworm is disclosed. Also disclosed is the entire nucleotide sequence of
a Cry5-like gene herein designated as Cry1I which was isolated from this
strain.
The Bacillus strain of the invention has a broad spectrum of activity. By
broad spectrum it is intended that the strains are active against insects
from more than one order, preferably against insects from several orders,
more preferably active against insects from at least three orders.
Additionally, the strains are active against noninsect pests. For purposes
of the present invention, pests include but are not limited to insects,
fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests
include insects selected from the orders Coleoptera, Diptera, Hymenoptera,
Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera,
Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc.,
particularly Coleoptera, Lepidoptera, and Diptera.
In one embodiment, the invention encompasses the Bt isolate known as
BtC-18, deposited Dec. 31, 1996 as ATCC Accession No. 55922 (American Type
Culture Collection, 10801 University Blvd., Manassas, Va.). Protein
toxins, and DNA which encodes the protein toxins are additionally
encompassed. The subject invention also includes variants of the Bt
isolate which have substantially the same pesticidal properties as the
exemplified isolate. These variants include mutants and recombinant
isolates. Procedures for making mutants are well known in the art and
include ultraviolet light and nitrosoguanidine.
The Bacillus thuringiensis isolate C-18 produces more than one kind of
crystal protein during sporulation. Microscopic examination of the
sporulated cells revealed at least three different shapes of crystals:
bipyrmidal similar to the lepidopteran-specific B. thuringiensis subsp.
kurstaki; circular or irregular as that produced by dipteran-specific B.
thuringiensis subsp. israelensis; and rhomboid-shaped similar to the
coleopteran-specific B. thuringiensis subsp. tenebrionis.
The presence of the different types of crystals indicated that the
bacterium can kill insects belonging to more than one order of insects.
Bioassays confirmed that conclusion. The spore-crystal complex of C-18
killed insects from at least three orders, Lepidoptera, Diptera, and
Coleoptera. Generally, Bt's produce only a single crystal and therefore
have limited insect activity. Of the Bt's which have been reported to kill
insects from two orders, the activity is not stable. In contrast, the
present Bt has been shown to be highly stable.
Of particular interest is the corn rootworm activity exhibited by the
Bacillus strains of the invention. Generally, Bt's have little or no
rootworm activity. In contrast, the present strain exhibits substantial
rootworm activity. By substantial activity is intended that the protein is
capable of killing the target insect when present in at least microgram
(.mu.g) quantities.
Methods are available in the art for the identification and isolation of
the protein or proteins associated with insecticidal activity, i.e.,
rootworm activity. Generally, proteins can be purified by conventional
chromatography, including gel-filtration, ion-exchange, and immunoaffinity
chromatography, by high-performance liquid chromatography, such as
reversed-phase high-performance liquid chromatography, ion-exchange
high-performance liquid chromatography, size-exclusion high-performance
liquid chromatography, high-performance chromatofocusing and hydrophobic
interaction chromatography, etc., by electrophoretic separation, such as
one-dimensional gel electrophoresis, two-dimensional gel electrophoresis,
etc. Such methods are known in the art. See for example, Ausubel et al.
(1988) Current Protocols in Molecular Biology, Vols. 1 & 2, (eds.) John
Wiley & Sons, NY.
Additionally, antibodies can be prepared against substantially pure
preparations of the protein. See, for example, Radka et al. (1983) J.
Immunol., 128:2804; and Radka et al. (1984) Immunogenetics, 19:63. Any
combination of methods may be utilized to purify protein having pesticidal
properties, particularly rootworm activity. As the protocol is being
formulated, insecticidal activity is determined after each purification
step to assure the presence of the toxin of interest.
Methods are available in the art to assay for insect activity. Generally,
the protein is mixed and used in feeding assays. See, for example Marrone
et al. (1985) J. of Economic Entomology, 78:290-293.
Such purification steps will result in a substantially purified protein
fraction. By "substantially purified" or "substantially pure" is intended
protein which is substantially free of any compound normally associated
with the protein in its natural state. "Substantially pure" preparations
of protein can be assessed by the absence of other detectable protein
bands following SDS-PAGE as determined visually or by densitometry
scanning. Alternatively, the absence of other amino-terminal sequences or
N-terminal residues in a purified preparation can indicate the level of
purity. Purity can be verified by rechromatography of "pure" preparations
showing the absence of other peaks by ion exchange, reverse phase or
capillary electrophoresis. The terms "substantially pure" or
"substantially purified" are not meant to exclude artificial or synthetic
mixtures of the proteins with other compounds. The terms are also not
meant to exclude the presence of minor impurities which do not interfere
with the biological activity of the protein, and which may be present, for
example, due to incomplete purification.
Once purified protein is isolated, the protein, or the polypeptides of
which it is comprised, can be characterized and sequenced by standard
methods known in the art. For example, the purified protein, or the
polypeptides of which it is comprised, may be fragmented as with cyanogen
bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C
endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem., 257:9751-9758; Liu
et al. (1983) Int. J. Pept. Protein Res., 21:209-215). The resulting
peptides are separated, preferably by HPLC, or by resolution of gels and
electroblotting onto PVDF membranes, and subjected to amino acid
sequencing. To accomplish this task, the peptides are preferably analyzed
by automated sequencers. It is recognized that N-terminal, C-terminal, or
internal amino acid sequences can be determined. From the amino acid
sequence of the purified protein, a nucleotide sequence can be synthesized
which can be used as a probe to aid in the isolation of the gene encoding
the pesticidal protein.
Similar protocols can be utilized to isolate nematode active proteins,
rootworm active proteins, or proteins having activity to other pests.
It is recognized that the pesticidal proteins may be oligomeric and will
vary in molecular weight, number of protomers, component peptides,
activity against particular pests, and in other characteristics. However,
by the methods set forth herein, proteins active against a variety of
insects or pests may be isolated and characterized.
Once the purified protein has been isolated and characterized it is
recognized that it may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for such
manipulations are generally known in the art. For example, amino acid
sequence variants of the pesticidal proteins can be prepared by mutations
in the DNA. Such variants will possess the desired pesticidal activity.
Obviously, the mutations that will be made in the DNA encoding the variant
must not place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary rnRNA structure.
See, EP Patent Application Publication No. 75,444.
In this manner, the present invention encompasses the active rootworm
proteins, and other insecticidal and pesticidal proteins, as well as
components and fragments thereof. That is, it is recognized that component
protomers, polypeptides or fragments of the proteins may be produced which
retain pesticidal activity. These fragments include truncated sequences,
as well as N-terminal, C-terminal, internal and internally deleted amino
acid sequences of the proteins.
Most deletions, insertions, and substitutions of the protein sequence are
not expected to produce radical changes in the characteristics of the
pesticidal protein. However, when it is difficult to predict the exact
effect of the substitution, deletion, or insertion in advance of doing so,
one skilled in the art will appreciate that the effect will be evaluated
by routine screening assays.
The proteins or other component polypeptides described herein may be used
alone or in combination. That is, several proteins may be used to control
different insect pests.
The pesticidal proteins of the invention can be used in combination with Bt
toxins or other insecticidal proteins to increase insect target range.
Other insecticidal principles include protease inhibitors (both serine and
cysteine types), lectins, .alpha.-amylase and peroxidase. This
co-expression of more than one insecticidal principle in the same
transgenic plant can be achieved by genetically engineering a plant to
contain and express all the genes necessary. Alternatively, separate
plants can be transformed with different components. By crossing the
plants, progeny are obtained which express all of the genes of interest.
It is recognized that there are alternative methods available to obtain the
nucleotide and amino acid sequences of the present proteins. For example,
to obtain the nucleotide sequence encoding the pesticidal protein, cosmid
clones, which express the pesticidal protein, can be isolated from a
genomic library. From larger active cosmid clones, smaller subclones can
be made and tested for activity. In this manner, clones which express an
active pesticidal protein can be sequenced to determine the nucleotide
sequence of the gene. Then, an amino acid sequence can be deduced for the
protein. For general molecular methods, see, for example, Molecular
Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al.
(eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989), and the references cited therein.
The present invention also encompasses nucleotide sequences from other
Bacillus strains and organisms other than Bacillus, where the nucleotide
sequences are isolatable by hybridization with the Bacillus nucleotide
sequences of the invention. Proteins encoded by such nucleotide sequences
can be tested for pesticidal activity. The invention also encompasses the
proteins encoded by the nucleotide sequences. Furthermore, the invention
encompasses proteins obtained from organisms other than Bacillus wherein
the protein cross-reacts with antibodies raised against the proteins of
the invention. Again the isolated proteins can be assayed for pesticidal
activity by the methods disclosed herein or others well-known in the art.
In this manner, the insecticidal genes of the present invention include
those coding for proteins homologous to, and having essentially the same
biological properties as, the insecticidal genes disclosed herein, and
particularly the rootworm active gene. This definition is intended to
encompass natural allelic variations in the genes. Cloned genes of the
present invention can be of other species of origin. Thus, DNAs which
hybridize to the present insecticidal genes are also an aspect of this
invention. Conditions which will permit other DNAs to hybridize to the DNA
disclosed herein can be determined in accordance with known techniques.
For example, hybridization of such sequences may be carried out under
conditions of reduced stringency, medium stringency or even stringent
conditions (e.g., conditions represented by a wash stringency of 35-40%
Formamide with 5.times. Denhardt's solution, 0.5% SDS and 1.times.SSPE at
37.degree. C.; conditions represented by a wash stringency of 40-45%
Formamide with 5.times.Denhardt's solution, 0.5% SDS, and 1.times.SSPE at
42.degree. C.; and conditions represented by a wash stringency of 50%
Formamide with 5.times.Denhardt's solution, 0.5% SDS and 1.times.SSPE at
42.degree. C., respectively, to DNA encoding the insecticidal genes
disclosed herein in a standard hybridization assay. See J. Sambrook et
al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring
Harbor Laboratory)). In general, sequences which code for insecticidal
protein and hybridize to the insecticidal gene disclosed herein will be at
least 75% homologous, 85% homologous, and even 95% homologous or more with
the sequences. Further, DNAs which code for insecticidal proteins, or
sequences which code for an insecticidal protein coded for by a sequence
which hybridizes to the DNAs which code for insecticidal genes disclosed
herein, but which differ in codon sequence from these due to the
degeneracy of the genetic code, are also an aspect of this invention. The
degeneracy of the genetic code, which allows different nucleic acid
sequences to code for the same protein or peptide, is well known in the
literature. See, e.g., U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2,
Table 1.
The hybridization probes may be cDNA fragments or oligonucleotides, and may
be labeled with a detectable group as discussed hereinbelow. Pairs of
probes which will serve as PCR primers for the insecticidal gene or a
protein thereof may be used in accordance with the process described in
U.S. Pat. Nos. 4,683,202 and 4,683,195.
Once the nucleotide sequences encoding the pesticidal proteins of the
invention have been isolated, they can be manipulated and used to express
the protein in a variety of hosts including other organisms, including
microorganisms and plants.
The pesticidal genes of the invention can be optimized for enhanced
expression in plants. See, for example, EPA 0359472; EPA 0385962; WO
91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328;
and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this
manner, the genes can be synthesized utilizing plant preferred codons.
That is the preferred codon for a particular host is the single codon
which most frequently encodes that amino acid in that host. The maize
preferred codon, for example, for a particular amino acid may be derived
from known gene sequences from maize. Maize codon usage for 28 genes from
maize plants is found in Murray et al. (1989) Nucleic Acids Research,
17:477-498, the disclosure of which is incorporated herein by reference.
Synthetic genes can also be made based on the distribution of codons a
particular host uses for a particular amino acid.
In this manner, the nucleotide sequences can be optimized for expression in
any plant. It is recognized that all or any part of the gene sequence may
be optimized or synthetic. That is, synthetic or partially optimized
sequences may also be used.
In like manner, the nucleotide sequences can be optimized for expression in
any microorganism. For Bacillus preferred codon usage, see, for example
U.S. Pat. No. 5,024,837 and Johansen et al. (1988) Gene, 65:293-304.
Methodologies for the construction of plant expression cassettes as well as
the introduction of foreign DNA into plants are described in the art. Such
expression cassettes may include promoters, terminators, enhancers, leader
sequences, introns and other regulatory sequences operably linked to the
pesticidal protein coding sequence.
Methodologies for the construction of plant expression cassettes are
described in the art. The construct may include any necessary regulators
such as terminators, (Guerineau et al. (1991) Mol. Gen. Genet.,
226:141-144; Proudfoot (1991) Cell, 64:671-674; Sanfacon et al. (1991)
Genes Dev., 5:141-149; Mogen et al. (1990) Plant Cell, 2:1261-1272; Munroe
et al. (1990) Gene, 91:151-158; Ballas et al. (1989) Nucleic Acids Res.,
17:7891-7903; Joshi et al. (1987) Nucleic Acid Res., 15:9627-9639); plant
translational consensus sequences (Joshi, C. P. (1987) Nucleic Acids
Research, 15:6643-6653), enhancers, introns (Luehrsen and Walbot (1991)
Mol. Gen. Genet., 225:81-93) and the like, operably linked to the
nucleotide sequence. It may be beneficial to include 5' leader sequences
in the expression cassette construct. Such leader sequences can act to
enhance translation. See, for example, (Elroy-Stein et al. (1989) PNAS
USA, 86:6126-6130), Allison et al. (1986), Macejak and Sarnow (1991)
Nature, 353:90-94, Jobling and Gehrke (1987) Nature, 325:622-625, Gallie
et al. (1989) Molecular Biology of RNA, pp. 237-256, Lommel et al. (1991)
Virology, 81:382-385, and Della-Cioppa et al. (1987) Plant Physiology,
84:965-968.
It is further recognized that the components of the expression cassette may
be modified to increase expression. For example, truncated sequences,
nucleotide substitutions or other modifications may be employed. See, for
example Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328;
Murray et al. (1989) Nucleic Acids Research, 17:477-498; and WO 91/16432.
For tissue specific expression, the nucleotide sequences of the invention
can be operably linked to tissue specific promoters.
Methods are available in the art for the introduction and stable
incorporation of the expression cassettes into plants. Suitable methods of
transforming plant cells include microinjection (Crossway et al. (1986)
Biotechniques, 4:320-334), electroporation (Riggs et al. (1986) Proc.
Natl. Acad. Sci. USA, 83:5602-5606, Agrobacterium mediated transformation
(Hinchee et al. (1988) Biotechnology, 6:915-921), direct gene transfer
(Paszkowski et al. (1984) EMBO J., 3:2717-2722), and ballistic particle
acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;
WO91/10725 and McCabe et al. (1988) Biotechnology, 6:923-926). Also see,
Weissinger et al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al.
(1987) Particulate Science and Technology, 5:27-37 (onion); Christou et
al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)
Bio/Technology, 6:923-926 (soybean); Datta et al. (1990) Biotechnology,
8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA,
85:4305-4309 (maize); Klein et al. (1988) Biotechnology, 6:559-563
(maize); WO91/10725 (maize); Klein et al. (1988) Plant Physiol.,
91:440-444 (maize); Fromm et al. (1990) Biotechnology, 8:833-839; and
Gordon-Kamm et al. (1990) Plant Cell, 2:603-618 (maize); Hooydaas-Van
Slogteren & Hooykaas (1984) Nature (London), 311:763-764; Bytebier et al.
(1987) Proc. Natl. Acad. Sci. USA, 84:5345-5349 (Liliaccae); De Wet et al.
(1985) In The Experimental Manipulation of Ovule Tissues, ed. G. P.
Chapman et al. pp. 197-209. Longman, N.Y. (pollen); Kaeppler et al. (1990)
Plant Cell Reports, 9:415-418; and Kaeppler et al. (1992) Theor. Appl.
Genet., 84:560-566 (whisker-mediated transformation); D'Halluin et al.
(1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993) Plant
Cell Reports, 12:250-255 and Christou and Ford (1995) Annals of Botany,
75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology, 14:745-750
(maize via Agrobacterium tumefaciens); all of which are herein
incorporated by reference.
Alternatively, the plant plastid can be transformed directly. Stable
transformation of plastids have been reported in higher plants, see, for
example, Svab et al. (1990) Proc. Nat'l. Acad. Sci. USA, 87:8526-8530;
Svab & Maliga (1993) Proc. Nat'l Acad. Sci. USA, 90:913-917; Staub &
Maliga (1993) EMBO J., 12:601-606. The method relies on particular gun
delivery of DNA containing a selectable marker and targeting of the DNA to
the plastid genome through homologous recombination. Additionally, plastid
transformation can be accomplished by trans-activation of a silent
plastid-borne transgene by tissue-specific expression of a nuclear-encoded
and plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci., USA, 91:7301-7305.
The cells which have been transformed may be grown into plants in
accordance with conventional ways. See, for example, McCormick et al.
(1986) Plant Cell Reports, 5:81-84 (1986). These plants may then be grown,
and either pollinated with the same transformed strain or different
strains, and the resulting hybrid having the desired phenotypic
characteristic identified. Two or more generations may be grown to ensure
that the subject phenotypic characteristic is stably maintained and
inherited and then seeds harvested to ensure the desired phenotype or
other property has been achieved.
The Bacillus strains of the invention may be used for protecting
agricultural crops and products from pests. Alternatively, a gene encoding
the pesticide may be introduced via a suitable vector into a microbial
host, and said host applied to the environment or plants or animals.
Microorganism hosts may be selected which are known to occupy the
"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana)
of one or more crops of interest. These microorganisms are selected so as
to be capable of successfully competing in the particular environment with
the wild-type microorganisms, provide for stable maintenance and
expression of the gene expressing the polypeptide pesticide, and,
desirably, provide for improved protection of the pesticide from
environmental degradation and inactivation.
A number of methods are available for introducing a gene expressing the
pesticidal protein into the microorganism host under conditions which
allow for stable maintenance and expression of the gene. For example,
expression cassettes can be constructed which include the DNA constructs
of interest operably linked with the transcriptional and translational
regulatory signals for expression of the DNA constructs, and a DNA
sequence homologous with a sequence in the host organism, whereby
integration will occur, and/or a replication system which is functional in
the host, whereby integration or stable maintenance will occur.
Transcriptional and translational regulatory signals include but are not
limited to promoter, transcriptional initiation start site, operators,
activators, enhancers, other regulatory elements, ribosomal binding sites,
an initiation codon, termination signals, and the like. See, for example,
U.S. Pat. No. 5,039,523; U.S. Pat. No. 4,853,331; EPO 0480762A2; Sambrook
et al. supra; Molecular Cloning, a Laboratory Manual, Maniatis et al.
(eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982);
Advanced Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1980); and the references cited
therein.
General methods for employing the strains of the invention in pesticide
control or in engineering other organisms as pesticidal agents are known
in the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.
The Bacillus strains of the invention or the microorganisms which have been
genetically altered to contain the pesticidal gene and protein may be used
for protecting agricultural crops and products from pests. In one aspect
of the invention, whole, i.e., unlysed, cells of a toxin
(pesticide)-producing organism are treated with reagents that prolong the
activity of the toxin produced in the cell when the cell is applied to the
environment of target pest(s).
Alternatively, the pesticides are produced by introducing a heterologous
gene into a cellular host. Expression of the heterologous gene results,
directly or indirectly, in the intracellular production and maintenance of
the pesticide. These cells are then treated under conditions that prolong
the activity of the toxin produced in the cell when the cell is applied to
the environment of target pest(s). The resulting product retains the
toxicity of the toxin. These naturally encapsulated pesticides may then be
formulated in accordance with conventional techniques for application to
the environment hosting a target pest, e.g., soil, water, and foliage of
plants. See, for example EPA 0192319, and the references cited therein.
The active ingredients of the present invention are normally applied in the
form of compositions and can be applied to the crop area or plant to be
treated, simultaneously or in succession, with other compounds. These
compounds can be both fertilizers or micronutrient donors or other
preparations that influence plant growth. They can also be selective
herbicides, insecticides, fungicides, bacteriocides, nematocides,
mollusocides or mixtures of several of these preparations, if desired,
together with further agriculturally acceptable carriers, surfactants or
application-promoting adjuvants customarily employed in the art of
formulation. Suitable carriers and adjuvants can be solid or liquid and
correspond to the substances ordinarily employed in formulation
technology, e.g. natural or regenerated mineral substances, solvents,
dispersants, wetting agents, tackifiers, binders or fertilizers.
Preferred methods of applying an active ingredient of the present invention
or an agrochemical composition of the present invention which contains at
least one of the pesticidal proteins produced by the bacterial strains of
the present invention are leaf application, seed coating and soil
application. The number of applications and the rate of application depend
on the intensity of infestation by the corresponding pest.
The following experiments are offered by way of illustration and not by way
of limitation.
EXPERIMENTAL
Introduction
Bacillus thuringiensis (Bt) strain C-18 is a gram-positive sporeforming
bacterium that produces parasporal crystals which have multiple toxicity
against three orders of insects: Lepidoptera, Coleoptera and Diptera as
well as to nematodes. C-18 is unique because of its capacity to kill such
a wide range of agriculturally and biomedically important pests. No other
Bt strain or isolate has been reported to have such a wide host range. The
vast majority of Bt's can kill insects belonging to only one order of
insects.
Isolation
C-18 was isolated in Egypt from dead pink bollworm larvae harvested from
cotton bolls grown in cotton fields in Egypt. Standard bacteriological
procedures were used to isolate gram-positive, spore forming bacteria. The
larvae were washed twice with sterile deionized water, transferred to
fresh sterile water (1 ml), macerated with a sterile glass-rod before
being subjected to a heat treatment (5 min. at 100.degree. C.). The heat
treatment killed all the vegetative and non-sporulating microbes. 100
.mu.l samples were then streaked onto LB-agar plates and incubated at
30.degree. C. overnight. Individual colonies were then picked, streaked
onto fresh sporulating medium and incubated at 30.degree. C. for 48 hours.
The resulting colonies were stained with endospore stain and examined
microscopically. All standard bacteriological techniques including
physiological and biochemical reactions were employed to determine the
identity of the isolates and only those which matched Bacillus
thuringiensis (Bt) were subjected to detailed analysis and evaluation.
Among the isolates selected for determining insecticidal and nematocidal
activities was C-18.
A unique aspect of the isolate C-18 was its ability to produce more than
one kind of protein crystal during sporulation. Microscopic examination of
the stained sporulated cells revealed at least three different shapes of
crystals: bipyrimidial similar to the lepidopteran-specific B.
thuringiensis subsp. kurstaki; circular or irregular as that produced by
B. thuringiensis subsp. israelensis; and, rhomboid-shaped similar to the
B. thuringiensis subsp. tenebrionis (FIGS. 1 A&B).
Insect & Nematode Bioassays
The insecticidal activity of C-18 was measured using bioassays involving a
variety of insects that represent the Lepidoptera, Coleoptera and Diptera
orders of insects. Initial insect bioassays were performed with whole
bacterial cells and included the raspberry silkworm Philosamia ricini
(Lepidopteran), the mosquito Culex pipiens (Dipteran), and the flour
beetle Tribollium spp. (Coleopteran). Nematode bioassays were done with
the tomato nematode Meloidogyne incognita. Positive results from these
bioassays Table 1 prompted a more comprehensive analysis of the
insecticidal and nematocidal activities of C-18 parasporal crystals. As
can be seen in Table 1, C-18 is effective against a broad spectrum of
insects. Also parasporal crystals purified from C-18 exhibited the same
nematocidal activity as the whole organism (FIG. 1).
A more comprehensive bioassay system using larger numbers of insect
representing the three orders mentioned above was then performed.
In Table 1, bioassays were divided into three groups. Group I contains the
results of the lepidopteran insects (cotton leafworm Spodoptera
littoralis, cotton bollworm Pectinophora gossypiella, tobacco hornworm
Manduca sexta, raspberry silk worm Philosamia ricini, and a corn borer
Sesamia cratica). Group II includes dipteran insects (mosquitoes Culex
pipiens, Aedes aegypti, Aedes albopictus, Aedes triseriatus, and a blue
tongue virus vector: Culicoides variipennis). Group III represents the
coleopteran insects (flour beetle Tribollium spp., Colorado potato beetle
Leptinotarsa decemlineata, western corn rootworm Diabortica virgifera and
southern corn rootworm D. undecimpuncata howardi).
The bacterium was also tested against the nematode Melodogyne incognita
(FIG. 3).
TABLE 1
Pesticidal Activities of an Egyptian Isolate
Bacillus thuringiensis (BtC-18)
INSECT BtC-18 BTK BTI BTT
Group I: Lepidopteran-insects
Spodoptera littotalis.sup.1 10.00 40.00 0.0 0.0
Pectinophera gossypiella.sup.1 0.45 40.00 0.0 0.0
Manduca sexta.sup.1 0.45 7.00 0.0 0.0
Philosamia ricini.sup.1 0.43 3.00 0.0 0.0
Sessemia cratia.sup.1 20.00 30.00 0.0 0.0
Group II: Dipteran-insects
Culex pipiens.sup.2 7.00 0.0 5.50 0.0
Aedes aegypti.sup.2 200.00 0.0 40.00 0.0
Aedes albopictus.sup.2 36.00 0.0 nd 0.0
Aedes triseriatus.sup.2 360.00 0.0 nd 0.0
Culicoides variipennis.sup.2 18.00 0.0 NO 0.0
Group III: Coleopteran-insects
Tribollium sp..sup.3 35.00 0.0 0.0 30.00
Colorado Potato Beetle.sup.1 50.00 0.0 0.0 25.00
Western Corn Rootworm.sup.1 350.00 0.0 0.0 8,000.00
Southern Corn Rootworm.sup.1 350.00 0.0 0.0 10,000.00
.sup.1 The LC50 is expressed in ng/cm..sup.2
.sup.2 The LC50 is expressed in ng/ml.
.sup.3 The LC50 is expressed in ng/g.
*BtC-18 also exhibited toxic activity against Nematodes.
Microscopic Examination of C-18
C-18 produces at least three morphological types of parasporal crystals
(FIG. 1). The three types or shapes are: (i) bipyrimidal--characteristic
of those crystals that are toxic to lepidopterans, (ii) rounded, amorphous
clusters--characteristic of dipteran-specific crystals, and (iii)
rhomboid--characteristic of coleopteran-specific crystals. Presence of
these three morphological crystal types correlate with the three kinds of
insecticidal activities normally associated singularly with all other Bt's
described in the scientific literature.
Profile of Proteins Extracted from Vegetative & Sporulating Cells of C-18
The molecular characterization of BtC-18 was determined at the protein and
the gene levels. Bacillus thuringiensis has two phases of growth, the
vegetative phase and the sporulation phase. The bacterium produces a
specific protein pool during each phase of growth. The proteins are the
expression products of active genes at the specific stage of development.
These proteins can be separated and visualized on a sodium
dedocylsulphate-polyacrylamide gel by electrophoresis (SDS-PAGE).
Proteins of vegetative and sporulating cells of the various strains of Bt
described in the literature have common and characteristic banding
patterns when analyzed by SDS-polyacrylamide gel electrophoresis. To
determine whether C-18 has a distinctive protein profile of its own,
proteins extracted from both vegetative and sporulating cells were
examined by this technique and compared with Bt subspecies kurstaki
(lepidopteran-specific), israeliensis (dipteran-specific), and
tenebrionsis (coleopteran-specific). C-18 does, indeed, have distinctive
protein profiles when compared with several other commonly known
subspecies of Bt.
Immunochemical Staining of Crystal Proteins
Western analysis of purified crystal proteins from C-18 was performed and
proteins identified by this technique were compared with the same three
subspecies of Bt mentioned above. Although there are some similarities
among the four organisms, C-18 does exhibit a distinctive and
characteristic crystal protein profile.
Plasmid DNA Profiles
Genes responsible for encoding insecticidal proteins that constitute
parasporal crystals of Bt usually are associated with plasmid DNA although
there is evidence that such genes are chromosomally linked as well. The
plasmids purified from C-18 are displayed in FIG. 2. The plasmid profile
of C-18 is distinctive when compared to the profiles of the same three
subspecies of Bt indicated above.
This bacterium contains a large number of plasmid DNA molecules. The
majority of the toxin genes have been reported to be carried on one of the
large plasmids, however, few have been reported on the chromosome of some
Bts. The plasmid profile of C-18 has been used as a tool to differentiate
between bacterial species. The plasmid profile of the BtC-18 was found to
be different from the other subspecies of B. thuringiensis tested as
indicated in FIG. 2.
Polymerase Chain Reaction (PCR)
To determine the existence of genes (Cry genes) in C-18 that may encode
different insecticidal proteins (Cry proteins), specific DNA primers were
constructed and used in PCR analysis of C-18 genomic DNA. The primers were
custom designed against conserved regions in the genomes of the same
subspecies used above. Amplification of various DNA fragments revealed
that C-18 contains similar Cry1 (Lepidopteran-specific) genes as Bt subsp.
kurstaki but different Cry genes that those of Bt subspecies israelensis
and tenebrionis (FIG. 4).
The proof of the existence or absence of the three genes encoding the three
crystals produced by BtC-18 was proven by the use of specific DNA primers
(three sets, each consisting of two pairs specific for one gene) designed
against conserved regions in the genomes of B. thuringiensis subsp.
kurstaki (Lep1A, Lep1B, Lep2A, and Lep2B), B. thuringiensis subsp.
israelensis (Dip1A, Dip1B, Dip2A, and Dip2B) and B. thuringiensis subsp.
tenebrionis (Col1A, Col1B, Col2A, and Col2B).
The primers were used in the polymerase chain reaction (PCR) to give
specific product profiles. The two pairs of Lep primers amplify a 0.49 kb
and a 0.908 kb DNA fragment. The Dip primers amplify a 0.797 kb and 1.290
kb DNA fragment. The Col primers amplify a 0.649 kb and 1.060 kb DNA
fragments (FIG. 4).
Western blot analysis of the crystal proteins produced by BtC-18 confirm
that the crystal proteins of BtC-18 are different from the subspecies
israelensis and tenebrionis.
Purification of the C18 Crystal Protein Complex and Insecticidal Properties
The BtC18 crystal complex was purified from the sporulated culture broth by
Renographin gradient from the methods discussed in Lee et al. (1995)
Biochem. Biophys. Res. Comm. 126: 953-960. The purified crystals were
washed several times with de-ionized water to get rid of any contaminants.
The crystal complex was subjected to differential solublization at various
pH's according to the methods of Dai and Gill (1993) Insect Biochem.
Molec. Biol. 23:273-283 and Hofte et al. (1986) Eur. J. Biochem.
161:273-280 and fractions (F1, F2, F3, F4, F5, and F6) obtained from this
procedure were bioassayed against corn rootworms (southern and western) as
representative of coleopteran insects and tobacco hornworm as lepidopteran
insects. All fractions killed both representative insects, but with
varying degrees. Fractions F2, F4, and F6 killed rootworms very
efficiently. 5 .mu.g/cm.sup.2 of the fractions killed 80-90% of the tested
insects. 100% kill was recorded at 10 .mu.g/cm.sup.2. All fractions killed
the tobacco hornworm albeit with variable efficiencies.
Binding of BtC18 Toxins to Specific Brush Border Membrane Proteins From the
Midgut of Different Insects
Brush border membrane vesicles (BBMV) were prepared from: corn rootworms
(WCRW and SCRW), tobacco hornworm (MS), and European corn borer (ECB)
according to methods used by Wolfersberger et al. (1987) Comp. Biochem.
Physiol. 86A:301-308. Specific amounts (20-MS, 50-ECB, and 120-W/SCRW
.mu.g/lane) of BBMV's were loaded and separated by SDS-PAGE. The separated
proteins were electro-blotted onto PVDF nylon membranes and were reacted
with 125 I labeled fractions (F2, F4, F5, and F6) from BtC18, which bind
to specific receptors from the BBMV. The radioactive membranes are then
exposed on film. Where there's a band there is an interaction with the
specific receptor. Evident in this analysis is the broad based interaction
of F6 radiolabelled proteins with the various brush border membrane
proteins extracted from these pests (Table 2). Based on this observation,
F6 appears to have the largest range of activity.
TABLE 2
BtC18
Protein BBMV Binding Protein (kDa)
fraction MS ECB SCRW WCRW
F2 210 nd nd nd
F4 210 nd nd nd
84 nd nd nd
45 nd nd nd
F5 210 nd nd nd
120 nd nd nd
60 nd nd nd
55 nd nd nd
F6 210 210 210 nd
120 84 nd 180
60 nd 150 150
45 nd 50 50
40 nd 42 42
Identification of Cry Genes
DNA primers available in the scientific literature enabled the
identification of the types of genes BtC-18 isolate. Some of the primers
were designed to differentiate between the members of the same family of
genes such as Cry1 family, Cry2 genes, Cry3 genes, Cry4 genes and Cry5
gene. These primers were used in PCR analysis to determine the number and
kinds of the different genes present in the isolate BtC-18. Several genes
were determined to be present in BtC-18. The identified genes were Cry1Aa,
Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry2A, Cry2B, and a Cry5-like gene
designated as Cry1I.
These genes can be grouped into three major families: i) Cry1 family genes
encode for proteins toxic to lepidopteran insects only, Cry1Aa, Cry1Ab,
Cry1Ac, Cry1C, Cry1D, ii) Cry2 genes encode proteins toxic to lepidopteran
and dipteran insects, Cry2A and Cry2B, and iii) Cry1B and Cry5 genes
encodes proteins toxic against lepidopterous and coleopterans insects. A
pool of at least nine genes were present in this single bacterium, which
covers the widest range of insecticidal activity recorded in the
scientific literature.
Identification of Additional Genes with Potential Insecticidal Activity
In the same manner as the above example, additional genes were discovered
to exist in BtC18 which are potentially responsible for giving this strain
its broad spectrum of insecticidal activity. Cry1F, Cry1G, Cry1K, and
Cry1M were found as well as nematode specific genes, designated nem1,
nem3, nem5 and nem7. In addition, a vegetative toxin was discovered in
BtC18 which gives a PCR generated band of the size expected for strain
BtHD1. This vegetative toxin is produced during vegetative growth and
kills black cut worm of corn (BCW).
Cloning of Genes
All the genes detected by PCR in BtC-18, mentioned above, were cloned into
BlueScript sk+II plasmid vector. Custom made DNA primers against the toxin
domains of the respective genes were used in PCR to amplify and target the
N-terminus part of the genes. The amplification products were eluted from
the gel, biocleaned and ligated to blunt ended pBlueScript. E. coli was
subsequently transformed with this vector, and, recombinants containing
the target genes were identified. The positive recombinants were
determined using the specific DNA primers and PCR to identify the expected
gene products. Recombinants clones were expressed in E. coli and the total
cellular proteins of the recombinants were analyzed by SDS-PAGE. For
better expression, the genes were cloned into an expression plasmid
pTrc99.
Cry1I Gene
Analysis of the Cry1I expression products by SDS-PAGE was performed. Two
proteins were produced, a 70 kDa protein and a smaller 58 to 60 kDa
protein which are not produced by E. coli transformed with pTrc99 alone.
The Cry1I gene was subcloned for the purpose of restriction mapping and
DNA sequencing. Three nucleotide sequences of three different segments of
the Cry1I gene from BtC-18 were obtained using the dideoxy chain
termination method (sequences not shown). The three segments were the 5'
and 3' ends of the gene as well as a middle segment. The nucleotide
sequence of these three segments of the Cry1I gene from BtC-18 were
compared using Blast server to nucleotide sequences of the published
sequences of several different Cry5 genes. By this method identities and
homologies of the nucleotide and amino acid sequences were determined. The
closest relative to the C18 Cry1I gene is that of entomocidus. C18 Cry1I
shares 97% nucleotide identity and 92% amino acid identity with the
entomodidus Cry5. The complete nucleotide sequence of the C18 Cry1I has
been obtained and is represented in FIG. 5.
Cry1I Activity Against Rootworm
The C18 Cry1I gene was inserted into the expression vector pTrc99 with IPTG
induction. E.coli strain BL21 was transformed with vector plus Cry1I
insert and empty vector as a negative control. Once the bacteria had grown
to the appropriate cell density, they were pelleted and resuspended in 11
ml of buffer (10 mM MgSO4, 0.3% Tween-20, 2 mM PMSF). The cellular
suspension was sonicated and protein extracted. The concentration of the
protein extract was 45 mg/ml. WCRW (Western Corn Rootworm) larvae were
challenged with various concentrations of the protein extract and
mortality scored after three days. Results of this assay demonstrates a
dramatic increase in WCRW mortality compared to the negative control at
doses as low as 2.3 .mu.g/cm.sup.2. At these levels mortality of the
larvae exposed to the extract containing Cry1Ip is up 55% compared to the
mortality of the larvae unexposed. At 11.8 .mu.g/cm.sup.2 the mortality is
up 80%. See Table 3. This is clear evidence that Cry1I is one of the
constituents in the BtC18 insecticidal arsenal that demonstrates
anti-rootworm activity.
TABLE 3
Est. Cry1I
dose
Extract vol. Dead (WCRW) Live (WCRW) % Mortality (.mu.g/cm.sup.2)
0.1 ml 6 2 75 2.3
0.5 ml 8 0 100 11.8
1.0 ml 8 0 100 22.3
2.0 ml 8 0 100 44.7
Control 3 12 20 0
Purification of Insecticidal Proteins from BtC-18
FPLC Separation of BtC-18 crystal proteins
Protein fractions from BtC-18 were dialyzed, solubilized and then purified
on a Mono Q column following the FPLC procedure recommended by the
manufacturer:
1. Portions of the fractions were loaded onto the column and eluted using
NaCl gradients.
2. Each individual fraction was bioassayed against different insects,
analyzed by SDS-PAGE, and ligand binding studies were performed using the
midguts of different insects to identify specific receptor proteins.
Isolation of Crystal Proteins from BtC-18.
The bacterium BtC-18 was cultured in liquid sporulation medium (T3-medium)
at 30.degree.-32.degree. C. and the crystal proteins were purified
according to Lee et al. (1985) Biochem. Biophys. Res. Commun., 126:953-960
using the following procedure:
1. The cells were grown to the spore stage. The crystal proteins were
released into the medium following autolysis of the cells.
2. The spore-crystal complexes were separated by centrifugation (10,000
rpm, for 10 min at 4 C). The complex was washed twice each with 1M
potassium chloride in deionized water.
3. The pellet was resuspended in distilled water and homogenized with a
Dounce homogenizer followed by centrifugation to separate the protein
complexes. This step was repeated two times.
4. The pellet (2-4 g) was suspended in a 68% Renografin gradient (9.6 ml of
2% Triton X-100 and 20.4 ml of Renografin). The mixture was homogenized
and subjected to sonication 10 times (20 pulses each time).
5. The mixture was centrifugated at 27,000 rpm for 15 hours. The viscous
top band on the gradient contained the crystal proteins. The purity of the
crystal proteins was checked by microscopic examination.
6. The crystal pellet was resuspended in 30 ml of 55% Renografin, 1% Triton
X-100, homogenized with a glass Dounce homogenizer and sonicated 10 times
as above.
7. The mixture was centrifugated again at 27,000 rpm for 15 h and the
crystal proteins were precipitated as a white pellet.
8. The supernatant was carefully removed and the pellet was washed three
times with distilled water before lyophilization.
Solubilization of the Crystal Proteins from BtC-18
Solubilization of the crystal proteins was accomplished according to the
methods of Dai et al. (1993) Insect Biochem. Mol. Biol., 23:273-283 and
Hofte et al. (1986) Eur. J. Biochem., 161:273-280.
1.50 .mu.g of the purified crystal proteins were dissolved in 5 ml of the
following solution:
Sodium carbonate (pH 9.5) 50 mM
Dithiothreitol (DTT) 10 mM
Phenylmethylsulfonyl Fluoride (PMSF) 5 mM
Ethylenediaminetetraacetate, disodium 10 mM
The mixture was incubated at 37.degree. C. for 80 min. with continuous
shaking.
2. The mixture was spun at 14 K rpm for 15 min. and the supernatant was
analyzed by SDS-PAGE.
3. The pellet was dissolved in the following solution:
Sodium hydroxide (pH12) 50 mM
Dithiothreitol (DTT) 10 mM
Phenylmethylsulfonyl fluoride (PMS) 5 mM
and incubated at 37 C/1 hour.
4. The supernatant was collected again by centrifugation at 14 k for 15
min.
5. The fractions were dialyzed against Tris-HCl (50 mM, pH9) containing 50
mM NaCl.
After purification, the proteins are assayed for activity against insects
of interest.
All publications and patent applications mentioned in the specification are
indicative of the level of skill of those skilled in the art to which this
invention pertains. All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be obvious that certain changes and modifications may be practiced
within the scope of the appended claims.
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